Field of the Invention
The present invention relates to a proteoliposome, a production method thereof, and a biochip.
Description of the Related Art
The post-genome era has come, and research and development in which proteins purified from a living body are used for various purposes have been actively carried out. Among them, membrane proteins have attracted attention as a new target of drug discovery.
A receptor protein which is a membrane protein exists in a biological membrane, plays an important role in intravital signal transduction, and its impairment of function is largely concerned with diseases. In recent years, approximately 50% of existing drugs and approximately 70% of drugs under research and development are said to target the membrane protein.
In addition, research in a new field called nanomedicine wherein molecules are used in a nanometer range under controlled activity for drug discovery, as typified by a drug delivery system (DDS), has appeared as well, and there is a case where it is actually used for a medical treatment in the United States.
More specifically, a nanometer-sized liposome is formed from a lipid bilayer membrane (hereinafter, may be simply referred to as a “lipid membrane”), a receptor protein is embedded in the surface of the liposome, and a drug is encapsulated therein. Consequently, a localized delivery by a molecule identification ability of the receptor protein can be realized. The drug in the proteoliposome (liposome in which membrane proteins are embedded) can be selectively administered to a target cell. This technique is expected to be applied not only to a treatment for lungs, eyes or skin, but also to a treatment for cancer, neurodegenerative diseases, or cardiovascular diseases.
For a quick diagnosis or in drug discovery, a biochip is used in which proteins or antibodies are immobilized on a substrate to observe the response by various methods such as a surface plasmon resonance method (SPR method). However, since detection/observation of conformational change in the protein is performed by holding proteins having activity with a number of molecules on a substrate, followed by detecting an average conformational change in proteins therein, it has been difficult to precisely examine the connection between the structure/conformational change of a single protein molecule and its function.
Furthermore, exploring the connection between the structure/conformational change and the function has been carried out by observing the structure and conformational change of a single protein molecule. For example, Nakagawa et al. have observed the structural differences of membrane proteins before and after stimulation by using an electron microscope. However, this method is an observation method carried out by scattering of a number of proteins on a substrate, and thus it is difficult to determine the orientation of the protein under observation. Therefore, it has been necessary to observe dozens of or tens of thousands of protein molecules and carry out image processing in order to know the orientation of the protein by analogy and determine the structure of the protein molecule. In addition, since proteins are frozen according to the method, it has not been possible to make an observation of proteins retaining their activity.
An atomic force microscope (AFM) is capable of observing a nanometer-sized object in a solution, and accordingly a protein retaining activity can be observed, one molecule at a time. Consequently, if a condition wherein membrane proteins are reconstituted in a lipid membrane is created so as to mimic that in vivo, it is believed that detailed knowledge related to the function of membrane proteins in an organism can be obtained by a single-molecule observation by AFM.
When a part of protein is immobilized on a substrate or a protein interacts with a substrate, it is thought that the protein does not change in the same manner as in vivo. In particular, since membrane proteins exhibit a function only in a state of being in a biological membrane, it is necessary to observe membrane proteins that being constituted in a lipid bilayer membrane mimicking an organism, so as to examine closely the connection between the structure and function.
As mentioned above, it is very important to reconstitute membrane proteins in a lipid membrane, particularly in the pharmacological or industrial application of membrane proteins.
Reconstitution of membrane proteins to a lipid membrane is carried out mainly for purposes such as (1) DDS using proteoliposomes, (2) two-dimensional crystallization of membrane proteins for X-ray structure analysis and (3) channel activity measurement by an electrophysiological method.
In (1), as many membrane proteins as possible are embedded in a liposome because it is believed that the greater the number of membrane proteins, the better the drug effect. However, even if a large number of membrane proteins are embedded in a liposome, they aggregate in a proteoliposome, and as a result, the number of membrane proteins which can fulfill the function effectively was considered to be small.
Also in (2), a large number of membrane proteins are embedded in a lipid membrane in a state where the proteins are aligned while being in contact with each other, so as to give a crystalline structure. Also in (3), the aggregation has not been a matter as long as there are membrane proteins having activity. That is, examination on reconstituting membrane proteins by dispersing at a predetermined interval without causing aggregation has not been practically carried out. Therefore, a biochip which is suitable for use in observation of a single membrane-protein molecule or the like has been demanded.
For a structural observation at the single molecular level, membrane proteins need to be oriented in the same orientation for reconstitution, in addition to their needing to be distanced at a predetermined interval without each being aggregated with the others. In addition, upon the spreading on a substrate, membrane proteins need to be placed at the center of a lipid domain (region including a lipid membrane) in order to perform an accurate structural observation by making the effect from surrounding lipid molecules uniform.
For all of those reasons, it is desirable to control the aggregation of membrane proteins to be constituted in a proteoliposome, and to spread membrane proteins at a predetermined interval on a substrate of a biochip by controlling the aggregation. Moreover, it is desirable to control the aggregation to reconstitute membrane proteins, from the viewpoint of reducing the amount of membrane proteins to be used since they are generically difficult to obtain or a valuable resource.